The E. coli genome is a circular duplex DNA molecule containing a single replication origin, oriC. DNA replication that starts at this site is initiated by the binding of DnaA protein to specific sequences termed DnaA boxes. A series of discrete events follow to establish the protein machinery at each replication fork for bidirectional replication fork movement. This process to produce two progeny DNA molecules is coordinated to cell growth and is regulated at the step of initiation of chromosomal DNA replication. The long term objectives of this research are to understand the process of initiation of E. coli chromosomal DNA replication at the biochemical level, and to determine how this process is regulated. Our recent results indicate that DnaA protein interacts directly with DnaB in its entry at oriC. Furthermore, DnaA protein plays an active role in the loading of DnaB onto the DNA. In the next funding period, we will pursue several aims to establish how an intermediate of the initiation process termed the prepriming complex is assembled and how these proteins interact. Specific aims are: i) to determine the stoichiometry of DnaA, DnaB and DnaC in the prepriming complex formed at oriC and to investigate possible mechanisms that control the entry of DnaB, ii) to identify where DnaB enters at oriC, iii) to identify specific amino acids of DnaA that are important for binding to DnaB, iv) to determine of the physical form of DnaB protein that interacts with DnaA protein, v) to identify the regions of DnaA protein involved in self-aggregation, vi) to identify and characterize other proteins that bind to DnaA protein as an affinity ligand, and vii) to identify amino acids of DnaC that are important for interaction with DnaB. These studies will provide further insight on the role of DnaA protein in the initiation process. Furthermore, the biochemical events in the initiation process in this model system may have similarities to the mechanism of initiation of chromosomal replication in other organisms.